Biooxidation of gold-bearing arsenopyrite concentrates, using acidophilic microbial communities, is among the largest commercial biohydrometallurgical processes. However, molecular mechanisms of microbial responses to sulfide raw materials have not been widely studied. The goal of this research was to gain insight into the defense strategies of the acidophilic bacterium Sulfobacillus thermotolerans, which dominates microbial communities functioning in industrial biooxidation processes at >35 °C, against the toxic effect of the high-arsenic gold-bearing sulfide concentrate. In addition to extreme metal resistance, this acidophile proved to be one of the most As-tolerant microorganisms. Comparative proteomic analysis indicated that 30 out of 33 differentially expressed proteins were upregulated in response to the ore concentrate, while the synthesis level of the functional proteins required for cell survival was not negatively affected. Despite a high level of cellular metal(loid) accumulation, no specific metal(loid)-resistant systems were regulated. Instead, several proteins involved in the metabolic pathways and stress response, including MBL fold metallo-hydrolase, sulfide:quinone oxidoreductase, and GroEL chaperonin, may play crucial roles in resistance to the sulfide ore concentrate and arsenic, in particular. This study provides the first data on the microbial responses to sulfide ore concentrates and advances our understanding of defense mechanisms against toxic compounds in acidophiles.